The long-range goals of our research program are to elucidate the contribution of the electronic structure of metal sites to their reactivity in biological electron transfer reactions. This proposal is focused to study the factors controlling electron transfer in cytochrome oxidases. Cytochrome c oxidases are integral membrane proteins that catalyze the reduction of oxygen to water, through a complex array of events that involve electron transfer coupled to proton translocation across the membrane. CuA is a binuclear copper center that is the primary electron acceptor in cytochrome oxidases. CuA accepts electrons from cytochrome c, and then transfers them in an intramolecular reaction. These two electron transfer steps take place with high efficiency, despite the low driving force of the redox reactions involved. Different spectroscopic approaches have suggested that the efficiency of CuA sites is due to the low reorganization energy and the high valence delocalization in the binuclear core. This proposal will explore: (1) the modulation of thermally accessible excited states in the electronic structure of CuA through weak metal-ligand interactions, that are expected to contribute to electron transfer rates, (2) the role of the different ligands in conserving the metal site structure, and (3) the molecular recognition between cytochrome c and a soluble subunit from the oxidases containing the CuA site. Paramagnetic NMR will be used as the central spectroscopic tool of this project. The Specific Aims of this application are: (1) To study the electronic structure of natural and engineered CuA sites by using multinuclear paramagnetic NMR spectroscopy. This will allow us to map the spin density on the different metal ligands and to assess the effects of H-bonds in the copper-sulfur interaction; (2) To study the effect of point mutations of axial and His ligands in the CuA-soluble fragment from T.thermophilus. We will especially study the effect of mutations on the energy of low lying excited states that might be relevant for electron transfer; and (3) To study the interaction between cytochrome c552 and the CuA-Soluble fragment of the ba3 oxidase from T.thermophilus by NMR spectroscopy. These studies are aimed to provide structural information to electron transfer studies perfomed by other group in this system.